Immunology and Cell Biology (2014), 1–7 & 2014 Australasian Society for Immunology Inc. All rights reserved 0818-9641/14 www.nature.com/icb
ORIGINAL ARTICLE
CD154 þ CD4 þ T-cell dependence for effective memory influenza virus-specific CD8 þ T-cell responses Matthew R Olson1, Shirley GK Seah2, Kathryn Edenborough1, Peter C Doherty1,3, Andrew M Lew2 and Stephen J Turner1 CD40–CD154 (CD40 ligand) interactions are essential for the efficient priming of CD8 þ cytotoxic T lymphocyte (CTL) responses. This is typically via CD4 þ CD154 þ T-cell-dependent ‘licensing’ of CD40 þ dendritic cells (DCs); however, DCs infected with influenza A virus (IAV) upregulate CD154 expression, thus enabling efficient CTL priming in the absence of CD4 þ T activation. Therefore, it is unclear whether CD4 T cells and DCs have redundant or unique roles in the priming of primary and secondary CTL responses after infection. Here we determine the precise cellular interactions involved in CD40–CD154 regulation of both primary and secondary IAV-specific CTL responses. Infection of both CD40 KO and CD154 KO mice resulted in diminished quantitative and qualitative CTL responses after both primary and secondary infection. Adoptive transfer of CD154 þ , but not CD154 KO, CD4 T cells into CD154 KO mice restored both primary and secondary IAV-specific CD8 T-cell responses. These data show that, although CD154 expression on CD4 T cells and other cell types (that is, DCs) may be redundant for the priming of primary CTL responses, CD154 expression by CD4 T cells is required for the priming memory CD8 T cells that are capable of fully responding to secondary infection. Immunology and Cell Biology advance online publication, 29 April 2014; doi:10.1038/icb.2014.28 Interactions between CD40 and its ligand CD154 are central to ensuring both effective primary and secondary CD8 þ cytotoxic T-lymphocyte (CTL) responses to infection. Activated CD4 þ T cells have a key role in the priming of optimal acute CD8 þ CTL responses and in the establishment of CTL memory.1–4 In the context of acute CTL responses, activated CD4 þ T cells that express CD154 ‘licence’ dendritic cells (DCs) via ligation of CD40,5 resulting in more efficient cross-presentation of antigen1,6,7 and production of IL-12 production that can serve as a third signal,8 both of which ensure the development of highly differentiated CTL responses.9 It has long been recognised that CD4 þ T-cell help is key for initiating the developmental programme that leads to the establishment of memory CD8 þ T cells. In particular, it has been proposed that IL-2 produced by activated CD4 T cells during an immune response acts to promote memory T-cell programming.10 However, a more recent study demonstrated that only when activated CD4 T cells could express CD154, needed to license DCs via CD40 ligation, could CD8 þ memory T cells be generated.11 What is not clear is whether the CD40–CD154 interactions necessary for both primary and secondary CTL responses involve the same or distinct cell–cell interactions. Several reports have now demonstrated that acute CTL response to bacterial and viral infections, including influenza A virus (IAV), can
be induced in the absence of CD4 þ T-cell help.2,4,12,13 In the case of acute IAV infection, the observed CD4 independence of primary virus-specific CTL responses has been explained by the ability of IAV infection to directly activate TLR7, and subsequently induce CD154 expression on DCs.14 These activated DCs are then able to directly prime CD40-expressing virus-specific CD8 T cells.14 Thus, a paradox is apparent with primary IAV-specific CTL responses readily primed via CD154–CD40 interactions imparted by DC–CTL contacts, rather than by CD4–DC-dependent licensing. However, whereas IAV infection is capable of inducing an acute CTL response in the absence of CD4 þ T-cell help, the establishment of effective memory CTL populations is deficient.13,15 In this study, we attempt to resolve this apparent paradox by determining the precise role of CD154 þ CD4 þ T cells in the establishment of optimal acute and memory CD8 þ T-cell responses in the context of the IAV infection model. Mice deficient for both CD40 (CD40 KO) and CD154 (CD154 KO) displayed diminished acute CTL responses induced by IAV infection. Importantly, adoptive transfer of virus-specific CD154 þ CD4 þ T cells into CD154 KO, but not CD40 KO, mice was able to restore both the magnitude and effector function of IAV-specific CTL responses. The ability of CD154 þ CD4 þ T cells to restore effective CTL responses in CD154 KO mice also correlated with improved viral clearance.
1Peter Doherty Institute for Infection and Immunity, Department of Microbiology and Immunology, University of Melbourne, Parkville, Victoria, Australia; 2Division of Immunology, Walter and Eliza Hall Institute of Medical Research, Parkville, Victoria, Australia and 3Department of Immunology, St Judes Childrens Research Hospital, Memphis, TN, USA Correspondence: Professor SJ Turner, Peter Doherty Institute for Infection and Immunity, Department of Microbiology and Immunology, University of Melbourne, Parkville, Victoria 3010, Australia. E-mail: [email protected] Received 3 February 2014; revised 23 March 2014; accepted 25 March 2014
CD154-dependent programming of flu memory T-cell responses MR Olson et al 2
Further, adoptive transfer of CD154 þ CD4 þ T cells into CD154 KO mice also restored the ability of memory CTL to be effectively recalled. This suggests that CD4 þ T cells, activated in response to IAV infection, are the major source of CD154, and hence ensure efficient programming of robust effector and memory virus-specific CTL responses. RESULTS Influenza-specific CD8 T-cell responses are limited in CD40- and CD154-deficient mice Both CD40 KO and CD154 KO mice exhibit decreased virus-specific CTL responses and delayed viral clearance in a number of model systems.16–18 To further investigate this phenomenon, wild-type (wt), CD40 KO, CD154 KO and anti-CD154 (clone MR1)-treated mice were infected with the recombinant IAV, A/HKx31-OVA323,19 and the splenic DbNP366- and DbPA224-specific CTL responses were enumerated 10 days after infection (Figures 1a and b). The magnitude of both the DbNP366- and DbPA224-specific CTL responses was decreased (Po0.05, Bfourfold) in CD40 KO, CD154 KO and antiCD154-treated mice, when compared with wt controls (Figures 1a and b). Thus, this supports earlier evidence that the CD40–CD154 nexus is key in determining optimal primary virus-specific CTL responses.18 Interestingly, the ability of DbPA224-specific, but not DbNP366-specific, CTL to simultaneously produce IFN-g and IL-2 was
Figure 1 CD154 expression on CD4 T cells is sufficient and necessary for the generation of full magnitude primary virus-specific CD8 T-cell responses in the absence of CD154 on other cell types. wt, CD40 KO, anti-CD154treated (MR1) and CD154 KO mice were given PBS (filled bars) or 1 106 OTII CD4 T cells (open bars) 1 day before intranasal infection with 104 PFU of A/HKx31-OVA323. Ten days after infection, spleens were harvested from mice and the total number of DbNP366-specific (a) and DbPA224-specific (b) CD8 T cells were enumerated by IFN-g intracellular cytokine staining (ICS). These data represent at least two individual experiments with at least seven mice per group. *Po0.05, significantly different as compared with wt mice that had received PBS; #Po0.05, significantly different as compared with wt mice that had received OTII CD4 T cells. Immunology and Cell Biology
also diminished in the CD40 KO, CD154 KO and anti-CD154-treated mice when compared with wt controls (Figures 2a and b, Po0.05). WT CD4 T-cell help rescues influenza-specific CD8 T-cell responses in CD154-deficient mice To better define the cellular interactions that direct CD40–CD154dependent priming of acute IAV-specific CTL responses, we adoptively transferred wt OTII TCR transgenic CD4 þ T cells into CD40 KO, CD154 KO or anti-CD154-treated mice infected with HKx31-OVA323 and tissues were harvested for analysis as described earlier. Adoptive transfer of wt OTII CD4 þ T cells restored DbNP366and DbPA224-specific CTL responses in CD154 KO mice, but not in CD40 KO or anti-CD154-treated mice (Figures 1a and b, Po0.05). Moreover, adoptive transfer of wt OTII CD4 þ T cells restored the capacity of DbPA224-specific CTL in CD154-deficient mice to produce IL-2 (Figure 2b), and rescued full IFN-g production in both DbNP366and DbPA224-specific CTL (Figures 2c and d, Po0.05). These data suggest that, in the absence of CD154 expression on APCs, CD154-expressing CD4 þ T cells are sufficient for optimal primary IAV-specific CD8 T-cell responses. It is possible that molecules, other than CD154 on the transferred CD4 T cells, may have a role in rescuing CD8 T-cell responses in CD154 KO mice. However, adoptive transfer of CD154 KO OTIIs into CD154 KO recipients did not rescue the magnitude or functional capacity of IAV-specific CTL (Figures 1 and 2). Thus, CD154 expression on activated CD4 þ T cells is both necessary and sufficient for promoting sustained CTL responses after IAV infection when CD154 expression is absent on other cells such as DCs. CD154-sufficient CD4 T-cell help enhances viral clearance in CD154 / recipients Given that CD40 and CD154 are required for optimal IAV-specific CD8 T-cell responses in the spleen, we investigated whether these interactions were also critical in generating robust pulmonary CTL responses that may impact viral clearance. In these studies, the lungs of naı¨ve wt or CD154 KO mice that received wt OTIIs or phosphatebuffered saline (PBS) were assayed for pulmonary influenza-specific CTL responses 6.5 days after infection with HKx31-OVA323. Although the magnitude of the DbNP366- and DbPA224-specific CTL responses in the lung were similar in wt mice, with or without adoptive transfer of OTII CD4 þ T cells (Figures 3a and b, P40.05), CD154 KO mice exhibited significantly reduced numbers of pulmonary DbNP366- and DbPA224-specific CTL (Figures 3a and b, Po0.05). Importantly, both the total number of tetramer and IFN-g-producing DbNP366- and DbPA224-specific CTL was fully restored to wt levels in CD154 KO mice that had received CD154 þ OTII CD4 T cells (Figures 3a–d). Moreover, the functional capacity of DbPA224-specific CTL, both in terms of the amount of IFN-g per cell (Figure 3e) and the proportion of TNF-a þ of IFN-g þ CTL, was restored in CD154 KO mice that received CD154 þ OTII CD4 þ T cells. Interestingly, whereas the proportion of IL-2 þ IAV-specific CTL was diminished in CD154 KO mice in the primary response, there was no difference in the MFI of IL-2 producers. To determine whether adoptive transfer of wt OTII CD4 þ T cells enhanced clearance of virus from the lungs of CD154 KO mice, we assessed viral titres in each group of mice after infection. First, both wt and CD154 KO mice that received adoptive transfer of OTII CD4 T cells exhibited similar (P40.05) numbers of OTII cells in the lungs at this time point, suggesting that these cells proliferated to a similar extent in both groups of mice (Figure 3g). Adoptive transfer of naive CD4 þ OTII T cells into wt mice reduced viral loads B26-fold
CD154-dependent programming of flu memory T-cell responses MR Olson et al 3
Figure 2 CD154 on CD4 T cells is necessary for optimal IFN-g and IL-2 production by IAV-specific CD8 T cells. Mice were treated with PBS (left side of panels a, b) or OTII cells (right side of panels a, b), infected with A/HKX31-OVA323, and spleens were harvested at day 10 post infection as per Figure 1. The capacity of DbNP366- (a) and DbPA224-specific (b) CD8 T cells to co-produce IFN-g and IL-2 was determined by ICS in each group of mice. *Po0.05, significantly different as compared with wt mice that had received PBS. #Po0.05, represents significantly different as compared with wt mice that had received OTII CD4 T cells. The capacity of DbNP366- (c) and DbPA224-specific (d) CD8 T cells to produce IFN-g, as measured by MFI, was determined by ICS. *Po0.05, significantly different as indicated. These data represent at least two individual experiments with at least seven mice per group.
Figure 3 Early pulmonary virus-specific CD8 T-cell responses and virus clearance in CD154 KO mice are rescued following transfer of CD154 þ OTII CD4 T-cell help. wt (filled bars) or CD154 KO mice (open bars) received PBS or 1 106 OTII CD4 T cells and were infected as per Figure 1. Lungs were obtained on day 6.5 after infection and assessed for DbNP366- (a) and Db PA224- (b) specific CD8 T-cell responses by MHC-I tetramer staining and by IFN-g ICS (c, d). The capacity of Db PA224-specific CD8 T cells to produce IFN-g (MFI, e) and to co-produce IFN-g and TNF-a (%, f) was assessed by ICS. (g) The total number of OTII CD4 T cells in the lungs of each group of mice was enumerated. (h) Lungs were also assessed for virus titres by plaque assay. These data represent three independent experiments with at least nine mice per group. *Po0.05, significantly different as compared with wt mice that had received PBS. #Po0.05, significantly different as compared with CD154 KO mice that had received PBS.
compared with PBS-treated mice (Figure 3h, Po0.05) at day 6.5 after infection, suggesting that virus-specific CD4 þ T cells may directly impact viral clearance. Further, PBS-treated CD154 KO mice
demonstrated delayed viral clearance compared with wt mice (Figure 3h, Po0.05). CD154 KO mice that received wt OTII CD4 þ T cells exhibited significantly lower viral titres (Figure 3h, Po0.05) as Immunology and Cell Biology
CD154-dependent programming of flu memory T-cell responses MR Olson et al 4
compared with PBS-treated CD154 KO mice. Importantly, the folddecrease in viral load in CD154 KO mice receiving wt OTII CD4 T cells (B100-fold) was greater than observed in wt mice that had also received OTII cells (B26-fold). Both sets of mice cleared viral infection by day 10 after infection (data not shown). These data suggest that the enhanced virus-specific CD8 T-cell response observed in the CD154 / mice that received wt CD4 þ T-cell help further expedited viral clearance over what was observed with OTII cells alone. Thus, CD154 expression on activated CD4 T cells is sufficient for enhanced pulmonary virus-specific CTL responses after IAV infection and contributes to efficient clearance of virus from CD154 KO lungs. CD154 þ CD4 T-cell help rescues robust secondary virus-specific CD8 T-cell responses in CD154-deficient mice Whereas CD4 T-cell help is not always necessary for the generation of primary influenza-specific CTL responses, it is required for robust recall responses after heterologous viral challenge.2,4,15 Although the above data suggest that CD154 þ CD4 T-cell help is capable of rescuing a mature primary virus-specific CTL in an otherwise CD154deficient environment, it remained unclear whether CD154 þ CD4 T-cell help was also capable of rescuing secondary CTL responses after viral challenge in a CD154-deficient environment. To address this point, we adoptively transferred wt or CD154 KO OTII CD4 T cells into either wt or CD154 KO hosts and infected mice as shown in Figure 1. Infected mice were rested for 45–60 days before intranasal challenge with A/PR8 influenza virus (lacking the OVA323 epitope) with CTL responses analysed 8 days after secondary infection. In these experiments, there were similar numbers of splenic DbNP366- and DbPA224-specific CTL in wt mice that had received OTII CD4 þ T cells as compared with PBS (Figures 4a–c). Importantly, there was a marked decrease in the number of DbNP366- and DbPA224specific CTL in the spleens of CD154 KO mice that had received PBS
as a control (Figures 4a–c, Po0.05) and a small reduction in their capacity to produce IFN-g, as measured by MFI, as compared with wt controls (Figures 4d and e, Po0.05). We next asked whether transfer of virus-specific wt CD4 T-cell help could rescue CD8 T-cell responses after secondary infection. CD154 KO mice that had received wt OTII CD4 T cells exhibited a significant increase in the number of splenic DbNP366- and DbPA224specific CTL as compared with CD154 KO mice that had been given PBS (Figures 4a–c, Po0.05). Furthermore, the numbers of DbNP366and DbPA224-specific CD8 þ T cells and capacity of these cells to produce IFN-g were virtually identical in wt and CD154 KO mice that had received wt OTII CD4 þ T cells at the time of priming (Figures 4b and c), suggesting a full rescue of the secondary virus-specific CTL response. As with the primary response, adoptive transfer of CD154 KO OTIIs into CD154 KO recipients at the time of priming failed to rescue the magnitude of the DbNP366- and DbPA224-specific secondary CTL responses (Figures 4a–c). Further, cells generated in these mice exhibited a distinct defect in their capacity to produce IFN-g after restimulation as compared with controls (Figures 4d and e, Po0.05). These data strongly suggest that the upregulation of CD154 on activated CD4 þ T cells is necessary for the priming of both optimal primary and secondary CD8 þ T-cell responses. DISCUSSION Generation of optimal primary CD8 T-cell responses after infection depends on a number of shared factors that are largely independent of the pathogen. These factors include ligation of the TCR (signal 1), costimulation (signal 2) and inflammatory signals (signal 3). Interestingly, the requirement for CD4 T-cell help to promote effective primary CTL responses is highly dependent on the nature of the challenge. In particular, primary CTL responses to acute viral and bacterial infections are not dependent on CD4 help.4,7,10,15 As a potential explanation of this phenomenon, it has been suggested that
Figure 4 CD154 þ CD4 þ T-cell help is sufficient for full virus-specific CD8 T-cell responses after secondary infection. wt (filled bars) and CD154 KO mice (open bars) received PBS or 2 106 OTII CD4 T cells and were infected as per Figure 1. Mice were challenged intranasally with 2 103 PFU of IAV/PR8 45 days after primary infection. Spleens were harvested 8 days later and assessed for the total number of DbNP366- (a, b) and DbPA224-specific (a, c) CD8 T cells by IFN-g ICS. The relative capacity of DbNP366- (d) and DbPA224-specific (e) cells to produce IFN-g was measured by ICS. *Po0.05, significant difference. These data represent two individual experiments with at least 6 mice per group. Immunology and Cell Biology
CD154-dependent programming of flu memory T-cell responses MR Olson et al 5
primary CTL responses can be primed via CD154–CD40 interactions imparted by DC-CTL contacts, rather than through CD4-DCdependent licensing.14 Our data further support the importance of CD40 and CD154 in the generation of primary IAV-specific CD8 T-cell responses, as mice with altered components of this system exhibit significantly impaired CTL responses (Figure 1). Further, adoptive transfer of wt CD4 þ T-cell help into CD154-deficient hosts restored optimal IAV-specific CD8 T-cell numbers after infection (Figure 1). Ballesteros-Tato and colleagues have recently shed new light on this phenomenon, demonstrating that CD154 expression on CD4 þ T cells is required to maintain DC activation and therefore sustain primary IAV-specific CTL responses, particularly at latter stages of infection.20 The fact that DbNP366-specific responses were independent of CD40 signalling in the absence of Foxp3-regulatory CD4 þ T cells (Tregs) suggests that CD154–CD40 T-cell–DC interactions serve to counteract early contraction of the primary CD8 þ T-cell response by inhibiting the suppressive effects of Tregs at later stages of infection. As such, this may provide an explanation for our observation that adoptive transfer of CD4 þ CD154 þ T cells could rescue primary IAV-specific CTL responses in CD154-deficient mice. Although there was no difference in the expression levels of CD80/86 and MHC II by DCs 3 days after infection between any of the groups (data not shown), it is tempting to speculate that the addition of exogenous OTII T cells helps maintain DC activation at later stages of infection. This possibility is currently under investigation. Interestingly, adoptive transfer of wt CD4 T cells improved the capacity of DbPA224-specific, but not DbNP366-specific, CTLs to produce IL-2 in CD154-deficient hosts (Figures 2 and 3). Why CD4 help differentially affects these specificities is unclear. We have previously shown that DbPA224-specific CTL exhibits an increased capacity for simultaneous cytokine production, including increased IL-2 production, when compared with the DbNP366-specific set.21 The greater functionality of DbPA224-specific, compared with DbNP366-specific, CTL has been correlated with higher T-cell receptor avidity for peptide-MHC.21 Thus, it is intriguing to speculate that CD40–CD154 interactions may preferentially contribute to the increased functionality of high avidity CTL populations. If this were the case, this would argue for a role of CD154–CD40 interactions in helping programme effective CTL responses, most likely via ‘licensing’ of DCs and subsequent provision of some secondary signal/s. This is currently under investigation. A level of redundancy of CD154 expression is apparent after IAV infection with both activated CD4 þ and DCs capable of CD154 expression and the potential to contribute to sustained CTL responses.14 These data beg the question, why does this level of redundancy between CD4 T cells and DCs exist during primary infection? One possible explanation for this redundancy may involve generating CTL responses to pathogens with different cell or tissue tropisms. Viruses with restricted tropism for epithelial cells or specific tissues (that is, RSV, HSV) would be unable to directly activate DCs by infection and allow for self-licensing. Further, a number of viruses (that is, vaccinia virus) that do infect DCs also have mechanisms that directly inhibit de novo protein synthesis and MHC expression on the cell surface. Thus, it is likely that even if these infected cells can self-licence they may not be able to induce proper CTL responses. In these scenarios, CD4 T cells would be required to license cross-presenting DCs for optimal induction of anti-viral CD8 T-cell responses. Previous work has demonstrated that IAV-infected DCs are potent inducers of virus-specific CD8 T-cell responses.22 However, IAV-infected DCs have a reduced capacity to cross-present
non-related IAV antigens to prime other CD8 þ T cell specificities.23 On the basis of these data, redundant expression of CD154 on DCs and CD4 T cells may ensure optimal CD8 T cells to de novo CTL responses in a situation where co-infection is prevalent. The above observations show a redundancy between CD154expressing CD4 T cells and other cell types that are capable of inducing primary IAV-specific CD8 T-cell responses. However, there is no apparent redundancy in the recall of memory IAV-specific CD8 T-cell responses after secondary infection. In virtually all situations, CD4 T cells are required for optimal secondary CD8 T-cell responses.2,3,7,15 This is especially evident in IAV infection, where MHCII-deficient mice have a blunted virus-specific secondary CD8 T-cell response.15 We also show here that secondary virus-specific CD8 T-cell responses are diminished in CD154-deficient mice (Figure 4). Like the primary response, secondary virus-specific CTL responses can also be rescued upon transfer of wt CD4 T-cell help. These data indicate that CD154 expression on CD4 T cells is sufficient for this response. Interestingly, while CD154 expression on DCs is sufficient for primary IAV-specific CTL responses,14 our data suggest that it is not sufficient for generation of memory CD8 T cells that can respond to secondary infection. In fact, utilising mixed bone marrow chimeras, we have recently demonstrated that CD154 expression on activated CD4s, but not DCs, is necessary for programming memory IAV-specific CD8 responses (Olson et al., manuscript under revision). This is also supported by the presence of CD154-sufficient DCs in MHCII or CD4-depleted/deficient mice and the lack of pathogenspecific secondary CD8 T-cell responses in these mice.3,15 Hence, programming of effective memory CTL populations likely occurs during the initial activation of naı¨ve, virus-specific CTL and supports the notion that CD40–CD154 interactions are key for establishing memory CTL populations capable of robust recall responses.11 We favour a model in which activated CD4s license DCs via a CD40L–CD40 interaction that promotes both robust primary and secondary CTL responses6,16,19 (Figure 5). Although CD154 signals from activated DCs can promote effector CTL differentiation, our data suggest that they cannot programme CTL memory formation. The implication is that CD40–CD154 interactions mediated by
Figure 5 Proposed model of how CD154 þ T-cell help restores both primary and secondary IAV CTL responses in CD154 / mice. Primary infection with the A/HKx31 (H3N2) IAV virus results in a diminished primary CTL response when compared with wild-type mice. Moreover, a lack of CD154 signals at the time of initial infection also led to poor memory CTL responses when challenged with a serologically distinct IAV (A/PR8, H1N1). Addition and subsequent activation after primary IAV infection of CD154 þ CD4 þ OTII T cells restores the primary CTL response of CD154-deficient animals. Moreover, CD154 / mice that received OTII help at the time of primary infection (OTII cells þ infection with A/HKX31-OVA323) are now able to mount robust secondary CTL responses after heterologous IAV challenge (A/PR8). The fact that this occurs in the absence of memory OTII activation suggests that CD4-dependent CD154–CD40 interactions at the time of primary infection are key for programming IAV-specific CTL memory responses. Immunology and Cell Biology
CD154-dependent programming of flu memory T-cell responses MR Olson et al 6
different cellular interactions may result in different transcriptional programmes within activated CTL. Thus, it will be of interest to define the transcriptional signatures of CTL that have received CD40–CD154 interactions from either CD4 þ T cells or DCs to delineate the precise molecular pathways that lead to full effector and memory CTL differentiation and we are currently following this line of investigation. METHODS Mice and viral infections Mice (C57BL/6J Ly5.2, CD40 KO Ly5.2, CD154 KO Ly5.2, OTII Ly5.1 and CD154 KO OTII Ly5.1) were bred and housed in specific pathogen-free conditions at the Department of Microbiology and Immunology at the University of Melbourne (Parkville, VIC, Australia) or at the Walter and Eliza Hall Institute (WEHI, Parkville, VIC, Australia). To examine primary CTL responses, mice were infected intranasally with 104 plaque-forming units (PFU) of the H3N2 strain A/HKx31, engineered to express the chicken ovalbumin 323–339 peptide within the hemagglutinin head.24 In some experiments, mice received 1 106 wt OTII CD4 T cells or 1 106 CD154 KO OTII CD4 T cells, which recognise the OVA323–339 peptide,25 isolated from LNs of OTII TCR transgenic mice 1 day before infection. Mice were treated with 200 ml of PBS or 50 mg of purified anti-CD154 antibody (MR1) 1 day before and 1 day after infection. In experiments in which we examined the secondary response to influenza virus infection, wt or CD154 KO mice received OTII CD4 T cells or PBS and were infected with A/HKx31-OVA323 as described above. These mice were rested for 45 days and subsequently infected with 2 103 PFU of A/Puerto Rico/8/34 (PR8; H1N1).
Tissue sampling and isolation of lymphocytes Spleens and LNs were harvested from mice and pressed between frosted glass slides to create a single cell suspension in Hank’s Balanced Salt Solution (HBSS, University of Melbourne Media Preparation Unit). Mononuclear cells were isolated by digestion of the lungs from infected mice with 1 mg ml 1 collagenase in HBSS for 30 min at 37 1C. Following digestion, lungs were pressed through a 70 mM nylon mesh filter and washed and resuspended in HBSS containing 10% fetal calf serum. Red blood cells were lysed in spleen and lung samples by treatment with ammonium tris chloride buffer (0.14 M NH4Cl, 0.017 M Tris) for 5 min at room temperature, followed by copious washing with HBSS.
MHC-I tetramer and antibody staining Mononuclear cells (1–2 106) from single cell suspensions were added to individual wells of a 96-well plate, pelleted by centrifugation (1600 r.p.m. for 6 min at 4 1C) and washed once in FACS buffer (PBS containing 1% BSA and 0.02% sodium azide). Following washing, cells were resuspended in 50 ml of FACS buffer containing DbNP366 and DbPA224 MHC-I tetramers (ImmunoID) conjugated to phycoerythrin (PE) and incubated for 1 h at room temperature. After this incubation, cells were washed once with FACS buffer and resuspended in an antibody cocktail containing anti-CD8 Pacific Blue (BD/Pharmingen, Nth Ryde, NSW, Australia) and CD44 phycoerythrin-Cy7 (eBioscience, San Diego, CA, USA) for 30 min at 4 1C followed by washing twice in FACS buffer. Flow cytometric analysis was performed on a FACS Canto II (BD/Pharmingen) and data were analysed using FlowJo v8.8.5 (Treestar Inc, Ashland, OR, USA) software.
Intracellular cytokine staining Mononuclear cells (1–2 106) from single cell preparations from spleens or lungs were added to individual wells of a 96-well plate and incubated for 5–6 h in the presence of 1 mg ml 1 GolgiPlug (BD) and 10 U ml 1 of human IL-2 (Roche, Dee Why, NSW, Australia) in the presence or absence of 1 mM NP366–374 (ASNENMETM) or PA224–223 (SSLENFRAYV) peptides (both from Auspep) at 37 1C and 5% CO2. After incubation, cells were stained for surface expression of CD8a and intracellular IFN-g (APC, eBioscience) and IL-2 (PE, eBioscience). Total numbers of IFN-g þ DbNP366- or DbPA224-specific CD8 T cells were determined by subtracting the frequency of IFN-g þ cells in unstimulated controls from those stimulated with peptide. Immunology and Cell Biology
Determination of lung viral titres Lungs from infected mice were harvested and washed immediately in cold HBSS. Lungs were placed in 2 ml of RPMI (Gibco Life Technologies, Mulgrave, VIC, Australia) containing 100 U ml 1 of penicillin and 24 mg ml 1 of gentamicin (both from Invitrogen, Carlsbad, CA, USA) and homogenised using a tissue homogeniser (Polytron 1200 PT). Lung homogenates were centrifuged for 10 min at 4 1C to pellet debris and supernatants were aliquoted and frozen at 80 1C for later viral titre determination on Madin–Darby canine kidney cells as previously described.26
Data analysis and statistics Data were analysed using Prism v4.0a software (GraphPad, La Jolla, CA, USA). Statistical analysis was carried out by Student’s t-test, or by ANOVA followed by a Tukey post-test where indicated. Differences were considered to be significant when Po0.05.
ACKNOWLEDGEMENTS We thank Drs Bridie Day and Brendan Russ for critical review of this manuscript. This work was supported by Australian National Health and Medical Research Council (NHMRC) program grant 5671222 (PCD and SJT) and 1037321 (AML), an NMHRC project grant 1043414 (AML), Juvenile Diabetes Research Foundation grant 112613, Victorian State Government Operational Infrastructure Support and Australian Government NHMRC IRIIS, DSO National Laboratories Scholarship, Singapore (SGKS), and an Australian Research Council Future Fellowship (SJT).
1 Bennett SR, Carbone FR, Karamalis F, Miller JF, Heath WR. Induction of a CD8 þ cytotoxic T lymphocyte response by cross-priming requires cognate CD4 þ T cell help. J Exp Med 1997; 186: 65–70. 2 Shedlock DJ, Shen H. Requirement for CD4 T cell help in generating functional CD8 T cell memory. Science 2003; 300: 337–339. 3 Smith CM, Wilson NS, Waithman J, Villadangos JA, Carbone FR, Heath WR et al. Cognate CD4( þ ) T cell licensing of dendritic cells in CD8( þ ) T cell immunity. Nat Immunol 2004; 5: 1143–1148. 4 Sun JC, Bevan MJ. Defective CD8 T cell memory following acute infection without CD4 T cell help. Science 2003; 300: 339–342. 5 Ridge JP, Di Rosa F, Matzinger P. A conditioned dendritic cell can be a temporal bridge between a CD4 þ T-helper and a T-killer cell. Nature 1998; 393: 474–478. 6 Bennett SR, Carbone FR, Karamalis F, Flavell RA, Miller JF, Heath WR. Help for cytotoxic-T-cell responses is mediated by CD40 signalling. Nature 1998; 393: 478–480. 7 Schoenberger SP, Toes RE, van der Voort EI, Offringa R, Melief CJ. T-cell help for cytotoxic T lymphocytes is mediated by CD40–CD40L interactions. Nature 1998; 393: 480–483. 8 Schulz O, Edwards AD, Schito M, Aliberti J, Manickasingham S, Sher A et al. CD40 triggering of heterodimeric IL-12 p70 production by dendritic cells in vivo requires a microbial priming signal. Immunity 2000; 13: 453–462. 9 Curtsinger JM, Lins DC, Mescher MF. Signal 3 determines tolerance versus full activation of naive CD8 T cells: dissociating proliferation and development of effector function. J Exp Med 2003; 197: 1141–1151. 10 Blachere NE, Morris HK, Braun D, Saklani H, Di Santo JP, Darnell RB et al. IL-2 is required for the activation of memory CD8 þ T cells via antigen cross-presentation. J Immunol 2006; 176: 7288–7300. 11 Feau S, Arens R, Togher S, Schoenberger SP. Autocrine IL-2 is required for secondary population expansion of CD8( þ ) memory T cells. Nat Immunol 2011; 12: 908–913. 12 Belz GT, Xie W, Altman JD, Doherty PC. A previously unrecognized H-2D(b)-restricted peptide prominent in the primary influenza A virus-specific CD8( þ ) T-cell response is much less apparent following secondary challenge. J Virol 2000; 74: 3486–3493. 13 Tripp RA, Sarawar SR, Doherty PC. Characteristics of the influenza virus-specific CD8 þ T cell response in mice homozygous for disruption of the H-2lAb gene. J Immunol 1995; 155: 2955–2959. 14 Johnson S, Zhan Y, Sutherland RM, Mount AM, Bedoui S, Brady JL et al. Selected Toll-like receptor ligands and viruses promote helper-independent cytotoxic T cell priming by upregulating CD40L on dendritic cells. Immunity 2009; 30: 218–227. 15 Belz GT, Wodarz D, Diaz G, Nowak MA, Doherty PC. Compromised influenza virus-specific CD8( þ )-T-cell memory in CD4( þ )-T-cell-deficient mice. J Virol 2002; 76: 12388–12393. 16 Borrow P, Tough DF, Eto D, Tishon A, Grewal IS, Sprent J et al. CD40 ligand-mediated interactions are involved in the generation of memory CD8( þ ) cytotoxic T lymphocytes (CTL) but are not required for the maintenance of CTL memory following virus infection. J Virol 1998; 72: 7440–7449.
CD154-dependent programming of flu memory T-cell responses MR Olson et al 7 17 Borrow P, Tishon A, Lee S, Xu J, Grewal IS, Oldstone MB et al. CD40L-deficient mice show deficits in antiviral immunity and have an impaired memory CD8 þ CTL response. J Exp Med 1996; 183: 2129–2142. 18 Lee BO, Hartson L, Randall TD. CD40-deficient, influenza-specific CD8 memory T cells develop and function normally in a CD40-sufficient environment. J Exp Med 2003; 198: 1759–1764. 19 Thomas PG, Brown SA, Morris MY, Yue W, So J, Reynolds C et al. Physiological numbers of CD4 þ T cells generate weak recall responses following influenza virus challenge. J Immunol 2010; 184: 1721–1727. 20 Ballesteros-Tato A, Leon B, Lund FE, Randall TD. CD4 þ T helper cells use CD154-CD40 interactions to counteract T reg cell-mediated suppression of CD8 þ T cell responses to influenza. J Exp Med 2013; 210: 1591–1601. 21 La Gruta NL, Turner SJ, Doherty PC. Hierarchies in cytokine expression profiles for acute and resolving influenza virus-specific CD8 þ T cell responses: correlation of cytokine profile and TCR avidity. J Immunol 2004; 172: 5553–5560.
22 Bhardwaj N, Bender A, Gonzalez N, Bui LK, Garrett MC, Steinman RM. Influenza virus-infected dendritic cells stimulate strong proliferative and cytolytic responses from human CD8 þ T cells. J Clin Invest 1994; 94: 797–807. 23 Smed-Sorensen A, Chalouni C, Chatterjee B, Cohn L, Blattmann P, Nakamura N et al. Influenza A virus infection of human primary dendritic cells impairs their ability to cross-present antigen to CD8 T cells. PLoS Pathog 2012; 8: e1002572. 24 Thomas PG, Brown SA, Morris MY, Yue W, So J, Reynolds C et al. Physiological numbers of CD4 þ T cells generate weak recall responses following influenza virus challenge. J Immunol 2010; 184: 1721–1727. 25 Barnden MJ, Allison J, Heath WR, Carbone FR. Defective TCR expression in transgenic mice constructed using cDNA-based alpha- and beta-chain genes under the control of heterologous regulatory elements. Immunol Cell Biol 1998; 76: 34–40. 26 Tannock GA, Paul JA, Barry RD. Relative immunogenicity of the cold-adapted influenza virus A/Ann Arbor/6/60 (A/AA/6/60-ca), recombinants of A/AA/6/60-ca, and parental strains with similar surface antigens. Infect Immunol 1984; 43: 457–462.
Immunology and Cell Biology
ORIGINAL ARTICLE
CD154 þ CD4 þ T-cell dependence for effective memory influenza virus-specific CD8 þ T-cell responses Matthew R Olson1, Shirley GK Seah2, Kathryn Edenborough1, Peter C Doherty1,3, Andrew M Lew2 and Stephen J Turner1 CD40–CD154 (CD40 ligand) interactions are essential for the efficient priming of CD8 þ cytotoxic T lymphocyte (CTL) responses. This is typically via CD4 þ CD154 þ T-cell-dependent ‘licensing’ of CD40 þ dendritic cells (DCs); however, DCs infected with influenza A virus (IAV) upregulate CD154 expression, thus enabling efficient CTL priming in the absence of CD4 þ T activation. Therefore, it is unclear whether CD4 T cells and DCs have redundant or unique roles in the priming of primary and secondary CTL responses after infection. Here we determine the precise cellular interactions involved in CD40–CD154 regulation of both primary and secondary IAV-specific CTL responses. Infection of both CD40 KO and CD154 KO mice resulted in diminished quantitative and qualitative CTL responses after both primary and secondary infection. Adoptive transfer of CD154 þ , but not CD154 KO, CD4 T cells into CD154 KO mice restored both primary and secondary IAV-specific CD8 T-cell responses. These data show that, although CD154 expression on CD4 T cells and other cell types (that is, DCs) may be redundant for the priming of primary CTL responses, CD154 expression by CD4 T cells is required for the priming memory CD8 T cells that are capable of fully responding to secondary infection. Immunology and Cell Biology advance online publication, 29 April 2014; doi:10.1038/icb.2014.28 Interactions between CD40 and its ligand CD154 are central to ensuring both effective primary and secondary CD8 þ cytotoxic T-lymphocyte (CTL) responses to infection. Activated CD4 þ T cells have a key role in the priming of optimal acute CD8 þ CTL responses and in the establishment of CTL memory.1–4 In the context of acute CTL responses, activated CD4 þ T cells that express CD154 ‘licence’ dendritic cells (DCs) via ligation of CD40,5 resulting in more efficient cross-presentation of antigen1,6,7 and production of IL-12 production that can serve as a third signal,8 both of which ensure the development of highly differentiated CTL responses.9 It has long been recognised that CD4 þ T-cell help is key for initiating the developmental programme that leads to the establishment of memory CD8 þ T cells. In particular, it has been proposed that IL-2 produced by activated CD4 T cells during an immune response acts to promote memory T-cell programming.10 However, a more recent study demonstrated that only when activated CD4 T cells could express CD154, needed to license DCs via CD40 ligation, could CD8 þ memory T cells be generated.11 What is not clear is whether the CD40–CD154 interactions necessary for both primary and secondary CTL responses involve the same or distinct cell–cell interactions. Several reports have now demonstrated that acute CTL response to bacterial and viral infections, including influenza A virus (IAV), can
be induced in the absence of CD4 þ T-cell help.2,4,12,13 In the case of acute IAV infection, the observed CD4 independence of primary virus-specific CTL responses has been explained by the ability of IAV infection to directly activate TLR7, and subsequently induce CD154 expression on DCs.14 These activated DCs are then able to directly prime CD40-expressing virus-specific CD8 T cells.14 Thus, a paradox is apparent with primary IAV-specific CTL responses readily primed via CD154–CD40 interactions imparted by DC–CTL contacts, rather than by CD4–DC-dependent licensing. However, whereas IAV infection is capable of inducing an acute CTL response in the absence of CD4 þ T-cell help, the establishment of effective memory CTL populations is deficient.13,15 In this study, we attempt to resolve this apparent paradox by determining the precise role of CD154 þ CD4 þ T cells in the establishment of optimal acute and memory CD8 þ T-cell responses in the context of the IAV infection model. Mice deficient for both CD40 (CD40 KO) and CD154 (CD154 KO) displayed diminished acute CTL responses induced by IAV infection. Importantly, adoptive transfer of virus-specific CD154 þ CD4 þ T cells into CD154 KO, but not CD40 KO, mice was able to restore both the magnitude and effector function of IAV-specific CTL responses. The ability of CD154 þ CD4 þ T cells to restore effective CTL responses in CD154 KO mice also correlated with improved viral clearance.
1Peter Doherty Institute for Infection and Immunity, Department of Microbiology and Immunology, University of Melbourne, Parkville, Victoria, Australia; 2Division of Immunology, Walter and Eliza Hall Institute of Medical Research, Parkville, Victoria, Australia and 3Department of Immunology, St Judes Childrens Research Hospital, Memphis, TN, USA Correspondence: Professor SJ Turner, Peter Doherty Institute for Infection and Immunity, Department of Microbiology and Immunology, University of Melbourne, Parkville, Victoria 3010, Australia. E-mail: [email protected] Received 3 February 2014; revised 23 March 2014; accepted 25 March 2014
CD154-dependent programming of flu memory T-cell responses MR Olson et al 2
Further, adoptive transfer of CD154 þ CD4 þ T cells into CD154 KO mice also restored the ability of memory CTL to be effectively recalled. This suggests that CD4 þ T cells, activated in response to IAV infection, are the major source of CD154, and hence ensure efficient programming of robust effector and memory virus-specific CTL responses. RESULTS Influenza-specific CD8 T-cell responses are limited in CD40- and CD154-deficient mice Both CD40 KO and CD154 KO mice exhibit decreased virus-specific CTL responses and delayed viral clearance in a number of model systems.16–18 To further investigate this phenomenon, wild-type (wt), CD40 KO, CD154 KO and anti-CD154 (clone MR1)-treated mice were infected with the recombinant IAV, A/HKx31-OVA323,19 and the splenic DbNP366- and DbPA224-specific CTL responses were enumerated 10 days after infection (Figures 1a and b). The magnitude of both the DbNP366- and DbPA224-specific CTL responses was decreased (Po0.05, Bfourfold) in CD40 KO, CD154 KO and antiCD154-treated mice, when compared with wt controls (Figures 1a and b). Thus, this supports earlier evidence that the CD40–CD154 nexus is key in determining optimal primary virus-specific CTL responses.18 Interestingly, the ability of DbPA224-specific, but not DbNP366-specific, CTL to simultaneously produce IFN-g and IL-2 was
Figure 1 CD154 expression on CD4 T cells is sufficient and necessary for the generation of full magnitude primary virus-specific CD8 T-cell responses in the absence of CD154 on other cell types. wt, CD40 KO, anti-CD154treated (MR1) and CD154 KO mice were given PBS (filled bars) or 1 106 OTII CD4 T cells (open bars) 1 day before intranasal infection with 104 PFU of A/HKx31-OVA323. Ten days after infection, spleens were harvested from mice and the total number of DbNP366-specific (a) and DbPA224-specific (b) CD8 T cells were enumerated by IFN-g intracellular cytokine staining (ICS). These data represent at least two individual experiments with at least seven mice per group. *Po0.05, significantly different as compared with wt mice that had received PBS; #Po0.05, significantly different as compared with wt mice that had received OTII CD4 T cells. Immunology and Cell Biology
also diminished in the CD40 KO, CD154 KO and anti-CD154-treated mice when compared with wt controls (Figures 2a and b, Po0.05). WT CD4 T-cell help rescues influenza-specific CD8 T-cell responses in CD154-deficient mice To better define the cellular interactions that direct CD40–CD154dependent priming of acute IAV-specific CTL responses, we adoptively transferred wt OTII TCR transgenic CD4 þ T cells into CD40 KO, CD154 KO or anti-CD154-treated mice infected with HKx31-OVA323 and tissues were harvested for analysis as described earlier. Adoptive transfer of wt OTII CD4 þ T cells restored DbNP366and DbPA224-specific CTL responses in CD154 KO mice, but not in CD40 KO or anti-CD154-treated mice (Figures 1a and b, Po0.05). Moreover, adoptive transfer of wt OTII CD4 þ T cells restored the capacity of DbPA224-specific CTL in CD154-deficient mice to produce IL-2 (Figure 2b), and rescued full IFN-g production in both DbNP366and DbPA224-specific CTL (Figures 2c and d, Po0.05). These data suggest that, in the absence of CD154 expression on APCs, CD154-expressing CD4 þ T cells are sufficient for optimal primary IAV-specific CD8 T-cell responses. It is possible that molecules, other than CD154 on the transferred CD4 T cells, may have a role in rescuing CD8 T-cell responses in CD154 KO mice. However, adoptive transfer of CD154 KO OTIIs into CD154 KO recipients did not rescue the magnitude or functional capacity of IAV-specific CTL (Figures 1 and 2). Thus, CD154 expression on activated CD4 þ T cells is both necessary and sufficient for promoting sustained CTL responses after IAV infection when CD154 expression is absent on other cells such as DCs. CD154-sufficient CD4 T-cell help enhances viral clearance in CD154 / recipients Given that CD40 and CD154 are required for optimal IAV-specific CD8 T-cell responses in the spleen, we investigated whether these interactions were also critical in generating robust pulmonary CTL responses that may impact viral clearance. In these studies, the lungs of naı¨ve wt or CD154 KO mice that received wt OTIIs or phosphatebuffered saline (PBS) were assayed for pulmonary influenza-specific CTL responses 6.5 days after infection with HKx31-OVA323. Although the magnitude of the DbNP366- and DbPA224-specific CTL responses in the lung were similar in wt mice, with or without adoptive transfer of OTII CD4 þ T cells (Figures 3a and b, P40.05), CD154 KO mice exhibited significantly reduced numbers of pulmonary DbNP366- and DbPA224-specific CTL (Figures 3a and b, Po0.05). Importantly, both the total number of tetramer and IFN-g-producing DbNP366- and DbPA224-specific CTL was fully restored to wt levels in CD154 KO mice that had received CD154 þ OTII CD4 T cells (Figures 3a–d). Moreover, the functional capacity of DbPA224-specific CTL, both in terms of the amount of IFN-g per cell (Figure 3e) and the proportion of TNF-a þ of IFN-g þ CTL, was restored in CD154 KO mice that received CD154 þ OTII CD4 þ T cells. Interestingly, whereas the proportion of IL-2 þ IAV-specific CTL was diminished in CD154 KO mice in the primary response, there was no difference in the MFI of IL-2 producers. To determine whether adoptive transfer of wt OTII CD4 þ T cells enhanced clearance of virus from the lungs of CD154 KO mice, we assessed viral titres in each group of mice after infection. First, both wt and CD154 KO mice that received adoptive transfer of OTII CD4 T cells exhibited similar (P40.05) numbers of OTII cells in the lungs at this time point, suggesting that these cells proliferated to a similar extent in both groups of mice (Figure 3g). Adoptive transfer of naive CD4 þ OTII T cells into wt mice reduced viral loads B26-fold
CD154-dependent programming of flu memory T-cell responses MR Olson et al 3
Figure 2 CD154 on CD4 T cells is necessary for optimal IFN-g and IL-2 production by IAV-specific CD8 T cells. Mice were treated with PBS (left side of panels a, b) or OTII cells (right side of panels a, b), infected with A/HKX31-OVA323, and spleens were harvested at day 10 post infection as per Figure 1. The capacity of DbNP366- (a) and DbPA224-specific (b) CD8 T cells to co-produce IFN-g and IL-2 was determined by ICS in each group of mice. *Po0.05, significantly different as compared with wt mice that had received PBS. #Po0.05, represents significantly different as compared with wt mice that had received OTII CD4 T cells. The capacity of DbNP366- (c) and DbPA224-specific (d) CD8 T cells to produce IFN-g, as measured by MFI, was determined by ICS. *Po0.05, significantly different as indicated. These data represent at least two individual experiments with at least seven mice per group.
Figure 3 Early pulmonary virus-specific CD8 T-cell responses and virus clearance in CD154 KO mice are rescued following transfer of CD154 þ OTII CD4 T-cell help. wt (filled bars) or CD154 KO mice (open bars) received PBS or 1 106 OTII CD4 T cells and were infected as per Figure 1. Lungs were obtained on day 6.5 after infection and assessed for DbNP366- (a) and Db PA224- (b) specific CD8 T-cell responses by MHC-I tetramer staining and by IFN-g ICS (c, d). The capacity of Db PA224-specific CD8 T cells to produce IFN-g (MFI, e) and to co-produce IFN-g and TNF-a (%, f) was assessed by ICS. (g) The total number of OTII CD4 T cells in the lungs of each group of mice was enumerated. (h) Lungs were also assessed for virus titres by plaque assay. These data represent three independent experiments with at least nine mice per group. *Po0.05, significantly different as compared with wt mice that had received PBS. #Po0.05, significantly different as compared with CD154 KO mice that had received PBS.
compared with PBS-treated mice (Figure 3h, Po0.05) at day 6.5 after infection, suggesting that virus-specific CD4 þ T cells may directly impact viral clearance. Further, PBS-treated CD154 KO mice
demonstrated delayed viral clearance compared with wt mice (Figure 3h, Po0.05). CD154 KO mice that received wt OTII CD4 þ T cells exhibited significantly lower viral titres (Figure 3h, Po0.05) as Immunology and Cell Biology
CD154-dependent programming of flu memory T-cell responses MR Olson et al 4
compared with PBS-treated CD154 KO mice. Importantly, the folddecrease in viral load in CD154 KO mice receiving wt OTII CD4 T cells (B100-fold) was greater than observed in wt mice that had also received OTII cells (B26-fold). Both sets of mice cleared viral infection by day 10 after infection (data not shown). These data suggest that the enhanced virus-specific CD8 T-cell response observed in the CD154 / mice that received wt CD4 þ T-cell help further expedited viral clearance over what was observed with OTII cells alone. Thus, CD154 expression on activated CD4 T cells is sufficient for enhanced pulmonary virus-specific CTL responses after IAV infection and contributes to efficient clearance of virus from CD154 KO lungs. CD154 þ CD4 T-cell help rescues robust secondary virus-specific CD8 T-cell responses in CD154-deficient mice Whereas CD4 T-cell help is not always necessary for the generation of primary influenza-specific CTL responses, it is required for robust recall responses after heterologous viral challenge.2,4,15 Although the above data suggest that CD154 þ CD4 T-cell help is capable of rescuing a mature primary virus-specific CTL in an otherwise CD154deficient environment, it remained unclear whether CD154 þ CD4 T-cell help was also capable of rescuing secondary CTL responses after viral challenge in a CD154-deficient environment. To address this point, we adoptively transferred wt or CD154 KO OTII CD4 T cells into either wt or CD154 KO hosts and infected mice as shown in Figure 1. Infected mice were rested for 45–60 days before intranasal challenge with A/PR8 influenza virus (lacking the OVA323 epitope) with CTL responses analysed 8 days after secondary infection. In these experiments, there were similar numbers of splenic DbNP366- and DbPA224-specific CTL in wt mice that had received OTII CD4 þ T cells as compared with PBS (Figures 4a–c). Importantly, there was a marked decrease in the number of DbNP366- and DbPA224specific CTL in the spleens of CD154 KO mice that had received PBS
as a control (Figures 4a–c, Po0.05) and a small reduction in their capacity to produce IFN-g, as measured by MFI, as compared with wt controls (Figures 4d and e, Po0.05). We next asked whether transfer of virus-specific wt CD4 T-cell help could rescue CD8 T-cell responses after secondary infection. CD154 KO mice that had received wt OTII CD4 T cells exhibited a significant increase in the number of splenic DbNP366- and DbPA224specific CTL as compared with CD154 KO mice that had been given PBS (Figures 4a–c, Po0.05). Furthermore, the numbers of DbNP366and DbPA224-specific CD8 þ T cells and capacity of these cells to produce IFN-g were virtually identical in wt and CD154 KO mice that had received wt OTII CD4 þ T cells at the time of priming (Figures 4b and c), suggesting a full rescue of the secondary virus-specific CTL response. As with the primary response, adoptive transfer of CD154 KO OTIIs into CD154 KO recipients at the time of priming failed to rescue the magnitude of the DbNP366- and DbPA224-specific secondary CTL responses (Figures 4a–c). Further, cells generated in these mice exhibited a distinct defect in their capacity to produce IFN-g after restimulation as compared with controls (Figures 4d and e, Po0.05). These data strongly suggest that the upregulation of CD154 on activated CD4 þ T cells is necessary for the priming of both optimal primary and secondary CD8 þ T-cell responses. DISCUSSION Generation of optimal primary CD8 T-cell responses after infection depends on a number of shared factors that are largely independent of the pathogen. These factors include ligation of the TCR (signal 1), costimulation (signal 2) and inflammatory signals (signal 3). Interestingly, the requirement for CD4 T-cell help to promote effective primary CTL responses is highly dependent on the nature of the challenge. In particular, primary CTL responses to acute viral and bacterial infections are not dependent on CD4 help.4,7,10,15 As a potential explanation of this phenomenon, it has been suggested that
Figure 4 CD154 þ CD4 þ T-cell help is sufficient for full virus-specific CD8 T-cell responses after secondary infection. wt (filled bars) and CD154 KO mice (open bars) received PBS or 2 106 OTII CD4 T cells and were infected as per Figure 1. Mice were challenged intranasally with 2 103 PFU of IAV/PR8 45 days after primary infection. Spleens were harvested 8 days later and assessed for the total number of DbNP366- (a, b) and DbPA224-specific (a, c) CD8 T cells by IFN-g ICS. The relative capacity of DbNP366- (d) and DbPA224-specific (e) cells to produce IFN-g was measured by ICS. *Po0.05, significant difference. These data represent two individual experiments with at least 6 mice per group. Immunology and Cell Biology
CD154-dependent programming of flu memory T-cell responses MR Olson et al 5
primary CTL responses can be primed via CD154–CD40 interactions imparted by DC-CTL contacts, rather than through CD4-DCdependent licensing.14 Our data further support the importance of CD40 and CD154 in the generation of primary IAV-specific CD8 T-cell responses, as mice with altered components of this system exhibit significantly impaired CTL responses (Figure 1). Further, adoptive transfer of wt CD4 þ T-cell help into CD154-deficient hosts restored optimal IAV-specific CD8 T-cell numbers after infection (Figure 1). Ballesteros-Tato and colleagues have recently shed new light on this phenomenon, demonstrating that CD154 expression on CD4 þ T cells is required to maintain DC activation and therefore sustain primary IAV-specific CTL responses, particularly at latter stages of infection.20 The fact that DbNP366-specific responses were independent of CD40 signalling in the absence of Foxp3-regulatory CD4 þ T cells (Tregs) suggests that CD154–CD40 T-cell–DC interactions serve to counteract early contraction of the primary CD8 þ T-cell response by inhibiting the suppressive effects of Tregs at later stages of infection. As such, this may provide an explanation for our observation that adoptive transfer of CD4 þ CD154 þ T cells could rescue primary IAV-specific CTL responses in CD154-deficient mice. Although there was no difference in the expression levels of CD80/86 and MHC II by DCs 3 days after infection between any of the groups (data not shown), it is tempting to speculate that the addition of exogenous OTII T cells helps maintain DC activation at later stages of infection. This possibility is currently under investigation. Interestingly, adoptive transfer of wt CD4 T cells improved the capacity of DbPA224-specific, but not DbNP366-specific, CTLs to produce IL-2 in CD154-deficient hosts (Figures 2 and 3). Why CD4 help differentially affects these specificities is unclear. We have previously shown that DbPA224-specific CTL exhibits an increased capacity for simultaneous cytokine production, including increased IL-2 production, when compared with the DbNP366-specific set.21 The greater functionality of DbPA224-specific, compared with DbNP366-specific, CTL has been correlated with higher T-cell receptor avidity for peptide-MHC.21 Thus, it is intriguing to speculate that CD40–CD154 interactions may preferentially contribute to the increased functionality of high avidity CTL populations. If this were the case, this would argue for a role of CD154–CD40 interactions in helping programme effective CTL responses, most likely via ‘licensing’ of DCs and subsequent provision of some secondary signal/s. This is currently under investigation. A level of redundancy of CD154 expression is apparent after IAV infection with both activated CD4 þ and DCs capable of CD154 expression and the potential to contribute to sustained CTL responses.14 These data beg the question, why does this level of redundancy between CD4 T cells and DCs exist during primary infection? One possible explanation for this redundancy may involve generating CTL responses to pathogens with different cell or tissue tropisms. Viruses with restricted tropism for epithelial cells or specific tissues (that is, RSV, HSV) would be unable to directly activate DCs by infection and allow for self-licensing. Further, a number of viruses (that is, vaccinia virus) that do infect DCs also have mechanisms that directly inhibit de novo protein synthesis and MHC expression on the cell surface. Thus, it is likely that even if these infected cells can self-licence they may not be able to induce proper CTL responses. In these scenarios, CD4 T cells would be required to license cross-presenting DCs for optimal induction of anti-viral CD8 T-cell responses. Previous work has demonstrated that IAV-infected DCs are potent inducers of virus-specific CD8 T-cell responses.22 However, IAV-infected DCs have a reduced capacity to cross-present
non-related IAV antigens to prime other CD8 þ T cell specificities.23 On the basis of these data, redundant expression of CD154 on DCs and CD4 T cells may ensure optimal CD8 T cells to de novo CTL responses in a situation where co-infection is prevalent. The above observations show a redundancy between CD154expressing CD4 T cells and other cell types that are capable of inducing primary IAV-specific CD8 T-cell responses. However, there is no apparent redundancy in the recall of memory IAV-specific CD8 T-cell responses after secondary infection. In virtually all situations, CD4 T cells are required for optimal secondary CD8 T-cell responses.2,3,7,15 This is especially evident in IAV infection, where MHCII-deficient mice have a blunted virus-specific secondary CD8 T-cell response.15 We also show here that secondary virus-specific CD8 T-cell responses are diminished in CD154-deficient mice (Figure 4). Like the primary response, secondary virus-specific CTL responses can also be rescued upon transfer of wt CD4 T-cell help. These data indicate that CD154 expression on CD4 T cells is sufficient for this response. Interestingly, while CD154 expression on DCs is sufficient for primary IAV-specific CTL responses,14 our data suggest that it is not sufficient for generation of memory CD8 T cells that can respond to secondary infection. In fact, utilising mixed bone marrow chimeras, we have recently demonstrated that CD154 expression on activated CD4s, but not DCs, is necessary for programming memory IAV-specific CD8 responses (Olson et al., manuscript under revision). This is also supported by the presence of CD154-sufficient DCs in MHCII or CD4-depleted/deficient mice and the lack of pathogenspecific secondary CD8 T-cell responses in these mice.3,15 Hence, programming of effective memory CTL populations likely occurs during the initial activation of naı¨ve, virus-specific CTL and supports the notion that CD40–CD154 interactions are key for establishing memory CTL populations capable of robust recall responses.11 We favour a model in which activated CD4s license DCs via a CD40L–CD40 interaction that promotes both robust primary and secondary CTL responses6,16,19 (Figure 5). Although CD154 signals from activated DCs can promote effector CTL differentiation, our data suggest that they cannot programme CTL memory formation. The implication is that CD40–CD154 interactions mediated by
Figure 5 Proposed model of how CD154 þ T-cell help restores both primary and secondary IAV CTL responses in CD154 / mice. Primary infection with the A/HKx31 (H3N2) IAV virus results in a diminished primary CTL response when compared with wild-type mice. Moreover, a lack of CD154 signals at the time of initial infection also led to poor memory CTL responses when challenged with a serologically distinct IAV (A/PR8, H1N1). Addition and subsequent activation after primary IAV infection of CD154 þ CD4 þ OTII T cells restores the primary CTL response of CD154-deficient animals. Moreover, CD154 / mice that received OTII help at the time of primary infection (OTII cells þ infection with A/HKX31-OVA323) are now able to mount robust secondary CTL responses after heterologous IAV challenge (A/PR8). The fact that this occurs in the absence of memory OTII activation suggests that CD4-dependent CD154–CD40 interactions at the time of primary infection are key for programming IAV-specific CTL memory responses. Immunology and Cell Biology
CD154-dependent programming of flu memory T-cell responses MR Olson et al 6
different cellular interactions may result in different transcriptional programmes within activated CTL. Thus, it will be of interest to define the transcriptional signatures of CTL that have received CD40–CD154 interactions from either CD4 þ T cells or DCs to delineate the precise molecular pathways that lead to full effector and memory CTL differentiation and we are currently following this line of investigation. METHODS Mice and viral infections Mice (C57BL/6J Ly5.2, CD40 KO Ly5.2, CD154 KO Ly5.2, OTII Ly5.1 and CD154 KO OTII Ly5.1) were bred and housed in specific pathogen-free conditions at the Department of Microbiology and Immunology at the University of Melbourne (Parkville, VIC, Australia) or at the Walter and Eliza Hall Institute (WEHI, Parkville, VIC, Australia). To examine primary CTL responses, mice were infected intranasally with 104 plaque-forming units (PFU) of the H3N2 strain A/HKx31, engineered to express the chicken ovalbumin 323–339 peptide within the hemagglutinin head.24 In some experiments, mice received 1 106 wt OTII CD4 T cells or 1 106 CD154 KO OTII CD4 T cells, which recognise the OVA323–339 peptide,25 isolated from LNs of OTII TCR transgenic mice 1 day before infection. Mice were treated with 200 ml of PBS or 50 mg of purified anti-CD154 antibody (MR1) 1 day before and 1 day after infection. In experiments in which we examined the secondary response to influenza virus infection, wt or CD154 KO mice received OTII CD4 T cells or PBS and were infected with A/HKx31-OVA323 as described above. These mice were rested for 45 days and subsequently infected with 2 103 PFU of A/Puerto Rico/8/34 (PR8; H1N1).
Tissue sampling and isolation of lymphocytes Spleens and LNs were harvested from mice and pressed between frosted glass slides to create a single cell suspension in Hank’s Balanced Salt Solution (HBSS, University of Melbourne Media Preparation Unit). Mononuclear cells were isolated by digestion of the lungs from infected mice with 1 mg ml 1 collagenase in HBSS for 30 min at 37 1C. Following digestion, lungs were pressed through a 70 mM nylon mesh filter and washed and resuspended in HBSS containing 10% fetal calf serum. Red blood cells were lysed in spleen and lung samples by treatment with ammonium tris chloride buffer (0.14 M NH4Cl, 0.017 M Tris) for 5 min at room temperature, followed by copious washing with HBSS.
MHC-I tetramer and antibody staining Mononuclear cells (1–2 106) from single cell suspensions were added to individual wells of a 96-well plate, pelleted by centrifugation (1600 r.p.m. for 6 min at 4 1C) and washed once in FACS buffer (PBS containing 1% BSA and 0.02% sodium azide). Following washing, cells were resuspended in 50 ml of FACS buffer containing DbNP366 and DbPA224 MHC-I tetramers (ImmunoID) conjugated to phycoerythrin (PE) and incubated for 1 h at room temperature. After this incubation, cells were washed once with FACS buffer and resuspended in an antibody cocktail containing anti-CD8 Pacific Blue (BD/Pharmingen, Nth Ryde, NSW, Australia) and CD44 phycoerythrin-Cy7 (eBioscience, San Diego, CA, USA) for 30 min at 4 1C followed by washing twice in FACS buffer. Flow cytometric analysis was performed on a FACS Canto II (BD/Pharmingen) and data were analysed using FlowJo v8.8.5 (Treestar Inc, Ashland, OR, USA) software.
Intracellular cytokine staining Mononuclear cells (1–2 106) from single cell preparations from spleens or lungs were added to individual wells of a 96-well plate and incubated for 5–6 h in the presence of 1 mg ml 1 GolgiPlug (BD) and 10 U ml 1 of human IL-2 (Roche, Dee Why, NSW, Australia) in the presence or absence of 1 mM NP366–374 (ASNENMETM) or PA224–223 (SSLENFRAYV) peptides (both from Auspep) at 37 1C and 5% CO2. After incubation, cells were stained for surface expression of CD8a and intracellular IFN-g (APC, eBioscience) and IL-2 (PE, eBioscience). Total numbers of IFN-g þ DbNP366- or DbPA224-specific CD8 T cells were determined by subtracting the frequency of IFN-g þ cells in unstimulated controls from those stimulated with peptide. Immunology and Cell Biology
Determination of lung viral titres Lungs from infected mice were harvested and washed immediately in cold HBSS. Lungs were placed in 2 ml of RPMI (Gibco Life Technologies, Mulgrave, VIC, Australia) containing 100 U ml 1 of penicillin and 24 mg ml 1 of gentamicin (both from Invitrogen, Carlsbad, CA, USA) and homogenised using a tissue homogeniser (Polytron 1200 PT). Lung homogenates were centrifuged for 10 min at 4 1C to pellet debris and supernatants were aliquoted and frozen at 80 1C for later viral titre determination on Madin–Darby canine kidney cells as previously described.26
Data analysis and statistics Data were analysed using Prism v4.0a software (GraphPad, La Jolla, CA, USA). Statistical analysis was carried out by Student’s t-test, or by ANOVA followed by a Tukey post-test where indicated. Differences were considered to be significant when Po0.05.
ACKNOWLEDGEMENTS We thank Drs Bridie Day and Brendan Russ for critical review of this manuscript. This work was supported by Australian National Health and Medical Research Council (NHMRC) program grant 5671222 (PCD and SJT) and 1037321 (AML), an NMHRC project grant 1043414 (AML), Juvenile Diabetes Research Foundation grant 112613, Victorian State Government Operational Infrastructure Support and Australian Government NHMRC IRIIS, DSO National Laboratories Scholarship, Singapore (SGKS), and an Australian Research Council Future Fellowship (SJT).
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